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Single-Molecule Localization Microscopy with the Fluorescence-Activating and Absorption-Shifting Tag (FAST) System.


ABSTRACT: We develop and employ the Fluorescence-Activating and absorption-Shifting Tag (FAST) system for super-resolution (SR) imaging and single-molecule tracking based on single-molecule localizations. The fast off rate of fluorogen binding, combined with its spatially well-separated labeling of the densely expressed FAST fusion proteins, allowed single-molecule measurements to be performed in both living and fixed cells. The well-separated fluorescence localization density was achieved by either reversibly controlling the fluorogen concentration or by irreversibly photobleaching the FAST-fluorogen complex. The experimentally determined resolution of 28 nm allowed us to resolve Ensconsin-labeled microtubules and to track single molecules in mitochondria. Our results demonstrate that FAST is well-suited for single-molecule localization microscopy (SMLM). The small size and the availability of spectrally distinct fluorogens present unique advantages of the FAST system as a potential orthogonal labeling strategy that could be applied in conjunction with existing super-resolution dyes and photoactivatable proteins in versatile imaging applications.

SUBMITTER: Smith EM 

PROVIDER: S-EPMC8608280 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Single-Molecule Localization Microscopy with the Fluorescence-Activating and Absorption-Shifting Tag (FAST) System.

Smith Elizabeth M EM   Gautier Arnaud A   Puchner Elias M EM  

ACS chemical biology 20190523 6


We develop and employ the Fluorescence-Activating and absorption-Shifting Tag (FAST) system for super-resolution (SR) imaging and single-molecule tracking based on single-molecule localizations. The fast off rate of fluorogen binding, combined with its spatially well-separated labeling of the densely expressed FAST fusion proteins, allowed single-molecule measurements to be performed in both living and fixed cells. The well-separated fluorescence localization density was achieved by either rever  ...[more]

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